Single-chain immunoglobulin binding fragments, or sFv, are made up of the heavy chain variable (V.sub.H) and light-chain variable (V.sub.L) domains joined together through a short linker peptide. The sFv protein carries the antigen binding site, which confers binding specificity on the molecule. These molecules have been found to have an extremely rapid plasma and whole-body clearance rate in mice and rhesus monkeys (see, e.g., Milenic et al., Cancer Res. 51:6363-6371 (1991)). The sFv shows more rapid tumor penetration and more even distribution throughout the tumor mass (Yokota et al., Cancer Res. 52:3402-3408 (1992)) than a corresponding chimeric murine-human immunoglobulin. Recent studies have also shown, however, that the sFv and Fab' forms have lower equilibrium association constants (K.sub.a) than the dimeric parental forms of the molecule; this, along with rapid clearance, has manifested itself in a lower percent injected dose per gram of sFv being deposited in a tumor site.
The sFv molecule per se, however, will not be sufficient for therapeutic use in its native (unconjugated) form. The cytolytic functions, such as ADCC and complement-dependent cytotoxicity, reside in the Fc region, which is exclusively made up of the constant-region domains of the heavy chain (C.sub.H domains). For a native antibody to be therapeutically effective against tumors it must, therefore, carry both antigen binding site and the Fc region. It has also been shown that N-linked glycosylation of Asn-297 within the C.sub.H 2 domain is critical for binding to the Fc receptor of the human effector cells and is necessary for ADCC activity (Tao et al., J. Immunol. 143:2595-2601 (1989), Dorai et al., Hybridoma 10:211-217 (1991) and Horan Hand et al., Cancer Immunol. Immunother. 35:165-174 (1992)). Fc-linked glycosyl residues are also implicated in complement fixation (Tao et al., J. Immunol. 143:2595-2601 (1989)). Since glycosylation of the Fc region is a characteristic of the eukaryotic system, an unconjugated antibody for therapeutic application must be produced in eukaryotic cells.
Transfection of eukaryotic cells remains highly inefficient, at best. It is all the more inefficient to develop a transfectant synthesizing a functional antibody molecule encoded by two separate genes. Currently, it is not feasible to carry out ex vivo introduction of two immunoglobulin genes simultaneously in a significant percentage of a cell population for reintroduction of the transfected cells into the host for genetic immunotherapy.
Monoclonal antibody (mAb) CC49, a murine IgG1, is a second-generation monoclonal of mAb B72.3 (Colcher et al., Proc. Natl. Acad. Sci. USA 78:3199-3203 (1981)), which reacts with the tumor-associated glycoprotein TAG-72 (Johnson et al., Cancer Res. 46:850-857 (1986)) expressed on a variety of carcinomas. Murine CC49 was developed by immunizing mice with TAG-72 purified by B72.3 affinity chromatography. Compared with B72.3, CC49 has a higher antigen binding affinity (Muraro et al., Cancer Res. 48:4588-4596 (1988)) and targets human colon carcinoma xenografts in mice more efficiently and reduces the growth of the xenograft with greater efficacy (Colcher et al., Cancer Res. 48:4597-4603 (1988) and Molinolo et al., Cancer Res. 50:1291-1298 (1990)). Chimeric B72.3 with a human .gamma.l constant region has been shown to efficiently mediate antibody-dependent cellular cytotoxicity (ADCC). Results from ongoing clinical trials suggest that murine CC49 is a useful clinical reagent for targeting human colorectal carcinoma lesions.
While the single-chain sFv form of CC49 has been shown to have important diagnostic use, what is needed in the art is a means to provide therapeutically useful single chain binding molecules of CC49 and other clinically useful antibodies. Quite surprisingly, the present invention fulfills this and other related needs.